DNA sequencing is one of the most important analytical methods in modern biotechnology. Detailed reviews on current sequencing technologies are provided in M. L. Metzker, Nature Reviews 2010, 11, 31, and C. W. Fuller et al., Nature Biotechnology 2009, 27, 1013.
A well-known sequencing method is the Sequencing-by-synthesis (SBS) method first described by R. Tsien (WO 91/06678). According to this method, the nucleoside triphosphates are reversibly blocked by a 3′-protecting group, in particular esters and ethers. Examples for esters are alkanoic esters like acetyl, phosphates and carbonates. The nucleoside triphosphate usually comprises a label at the base.
A method of enzymatically synthesizing a polynucleotide of a predetermined sequence in a stepwise manner using reversibly 3′-blocked nucleoside triphosphates was described by Hiatt and Rose (U.S. Pat. No. 5,990,300). They disclose besides esters, ethers, carbonitriles, phosphates, phosphoramides, carbonates, carbamates, borates, sugars, phosphoramidates, phenylsulfenates, sulfates and sulfones also nitrates as reversible 3′-protecting group. The deprotection may be carried out by chemical or enzymatic means. There are neither synthesis procedures nor deprotection conditions and enzymatic incorporation data disclosed for the nitrate group. The claimed deblocking solution preferably contains divalent cations like Co2+ and a biological buffer like Tris. 3′-Blocked nucleoside triphosphates containing a label are not disclosed.
Buzby (US 2007/0117104) discloses nucleoside triphosphates for SBS which are reversibly protected at the 3′-hydroxyl group and carry a label at the base. The label is connected via a cleavable linker such as a disulfide linker or a photocleavable linker. The linker consists of up to about 25 atoms. 3′-Protection group can be besides hydroxylamines, aldehydes, allylamines, alkenes, alkynes, alcohols, amines, aryls, esters, ethers, carbonitriles, phosphates, carbonates, carbamates, borates, sugars, phosphoramidates, phenylsulfanates, sulfates, sulfones and heterocycles also nitrates.
There is no disclosure on the synthesis of nitrate protected nucleotides, the deprotection conditions or enzymatic incorporation.
Reversibly blocked nucleotides are also described by Milton et al. (US 2007/0166705). Protecting groups are based on acetalic structures or acetal precursors. One preferred structure is azidomethyl which has been commercialized by Illumina. The modified nucleotides also include a label attached via a cleavable linker such as acid-labile, disulfide or photolabile structures.
These linker structures are also described by Milton et al. in US 2006/0160081. Acid-labile acetal-type or acetal-precursor type linkers as well as photolabile urethane bonded linkers are disclosed. Preferable deprotection reagents are phosphines like TCEP. For the attachment of the cleavable linker to the nucleobase an aminopropargyl residue is preferably used.
Barnes et al. (U.S. Pat. No. 7,057,026 B2) discloses that 3′-protecting group and label are cleaved under identical conditions.
The synthesis of nitrate modified nucleosides have been described for instance by R. Boschan et al., Chemical Reviews 1955, 55, 485, J. Honeyman et al., Sugar Nitrates in Advances in Carbohydrate Chemistry, ed. by M. L. Wolfromm and R. S. Tipson, Academic Press, New York, 1957, 12, 117-135, E. Naimi et al., J. Med. Chem. 2003, 46, 995, G. H. Hakimelahi et al., Helv. Chim. Acta 1984, 67, 906, J. Giziewicz et al., J. Org. Chem. 1999, 64, 2149, G.-F. Huang et al., J. Org. Chem. 1977, 42, 3821 or in DE 2160104 and in DE 2606532.
Deprotection conditions of the nitrate protecting group are reviewed by R. Boschan et al., Chemical Reviews 1955, 55, 485, J. Honeyman et al., Sugar Nitrates in Advances in Carbohydrate Chemistry, ed. by M. L. Wolfromm and R. S. Tipson, Academic Press, New York, 1957, 12, 117-135 or by T. W. Greene, Protective groups in organic synthesis, Wiley 1981, page 70-71. Nitrate esters can be cleaved by solvolytic decomposition via hydrolysis using alkaline conditions or sulfuric acid or by reductive conditions via electrolytic reduction, Grignard reagents, hydrazines, hydrogenolysis, lithium aluminium hydride, metal-acid mixtures, metals or anorganic sulfides like hydrogen sulfide and ammonium sulfide, ammonium hydrogen sulfide or sodium sulfide. Cleavage by irradiation is also described.
Organic sulfides are not described as deprotection reagent for nitrate esters. However, R. A. Yeates et al. (Molecular Pharmacology 1985, 28, 555) describe the reaction between organic nitrates and organic sulfhydryl compounds in the context of mechanism of action of nitroglycerin, only weakly concentrated sulfhydryl compound solutions were used. A similar context is described in M. Feelisch et al., European Heart Journal 1988, 9 (Suppl. A), 57-62.
B. Canard et al. (PNAS 1995, 92, 10859-10863) describe that 3′-esterified 2′-deoxynucleoside 5′-triphosphates are false chain-terminator substrates since DNA polymerases can incorporate them into DNA and subsequently use this new 3′-end to insert the next correctly paired dNTP after partial enzymatic cleavage of the ester group.
Addition of pyrophosphatase to polymerase catalyzed DNA synthesis reactions to eliminate pyrophosphate and improve DNA synthesis has been described in WO 94/05797 and US 2003/0134276. Its use in sequencing reactions is also described therein.
In US 2008/0138804 a composition comprising a mutated polymerase, manganese or magnesium salts, an organic solvent and an inorganic pyrophosphatase are disclosed in the context of sequencing.
Addition of Mn(II) ions to the Therminator II buffer for the incorporation of 3′-O-azidomethyl and 3′-O-allyl protected nucleoside triphosphates is described in J. Guo et al., PNAS 2008, 105, 9145-9150) and T. S. Kim et al., ChemBioChem 2010, 11, 75-78.
However, all these methods are sub-optimal with respect to performance with respect to quantitative enzymatic incorporation of the 3′-reversibly blocked nucleotide and fast and efficient deprotection as well as effective blockage property of the reversible 3′-protecting group. Due to residual esterase activities of DNA polymerases, the 3′-ester derived blockages known in the art are never 100% complete (Rasolonjatovo and Sarfati, Nuclosides & Nucleotides, 18 (4&5), 1021-1022, 1999).
Thus, there is a need in the art for a solution to provide deoxynucleosides which comprise a reversible but completely blocked 3′ terminus.